US6281193B1 - Compounds that inhibit the binding of RAF-1 or 14-3-3 proteins to the beta chain of IL-2 receptor, and pharmaceutical compositions containing same - Google Patents

Compounds that inhibit the binding of RAF-1 or 14-3-3 proteins to the beta chain of IL-2 receptor, and pharmaceutical compositions containing same Download PDF

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US6281193B1
US6281193B1 US09/194,145 US19414599A US6281193B1 US 6281193 B1 US6281193 B1 US 6281193B1 US 19414599 A US19414599 A US 19414599A US 6281193 B1 US6281193 B1 US 6281193B1
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Terry Strom
Wlodzimierz Maslinski
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Applied Research Systems ARS Holding NV
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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00—Drug, bio-affecting and body treating compositions
    • Y10S424/81—Drug, bio-affecting and body treating compositions involving autoimmunity, allergy, immediate hypersensitivity, delayed hypersensitivity, immunosuppression, immunotolerance, or anergy

Definitions

  • the present invention concerns compounds such as proteins, peptides and organic compounds which are characterized by their ability to block the interaction between Raf-1 protein and/or 14-3-3 proteins with the intracellular domain of the ⁇ chain of the interleukin-2 receptor molecule (IL-2R ⁇ ), and thereby block the intracellular signaling process mediated by IL-2R ⁇ .
  • the compounds of the invention are intended to inhibit the activity of IL-2 or IL-15 where desired, for example in autoimmune diseases in general, or graft-versus-host reactions in particular.
  • the present invention also concerns in vitro assays for the isolation, identification and characterization of the above compounds, as well as pharmaceutical compositions containing as active ingredient one or more compounds of the invention.
  • Interleukin-2 is a T-cell derived factor that amplifies the response of T cells to any antigen by stimulating the growth of the T cells.
  • IL-2 is a critical T-cell growth factor which plays a major role in the proliferation of T cells that occurs subsequent to antigen activation, this proliferation resulting in the amplification of the number of T cells responsive to any particular antigen.
  • IL-15 can generally substitute for IL-2 to exert most, if not all, of these activities (Bamford et al., 1994).
  • the high affinity (Kd:10 ⁇ 11 M) IL-2 receptor (IL-2R) is composed of at least three non-covalently associated IL-2 binding proteins: the low affinity (Kd:10 ⁇ 8 M) p55 ( ⁇ chain) and the intermediate affinity subunits (Kd:10 ⁇ 9 M) p75 ( ⁇ chain) and p64 ( ⁇ chain) (Smith, K. A., 1988; Waldmann, T. A., 1993).
  • Proliferative signals for the T cells are delivered through high affinity IL-2 receptors consisting of all three subunits, but not via the low affinity site (Robb, R. J. et al., 1984; Siegal, J. P. et al., 1987; Hatakeyama, M. et al., 1989).
  • IL-2R ⁇ , IL-2R ⁇ , and IL-2R ⁇ chains have 13, 286 and 86 amino acid intracytoplasmic domains, respectively.
  • IL-15 a cytokine with many IL-2-like activities, also utilizes the IL-2R ⁇ as a part of its receptor complex (Giri et al., 1994).
  • This IL-2R ⁇ dependent signaling process is fundamental to the cellular effects induced by the binding of IL-2 to its receptor (IL-2R) as well as the effects induced by the binding of IL-15 to its receptor.
  • the IL-2R ⁇ and ⁇ chains, but not the ⁇ chain, are essential for IL-2- as well as IL-15-mediated signal transduction (Nakamura, Y. et al., 1994).
  • the 64 kDa IL-2R ⁇ chain protein is rapidly phosphorylated on tyrosine residues after stimulation with IL-2.
  • the ⁇ chain has also been shown to be a part of other receptor complexes such as the receptor for IL-4 and IL-7 (Noguchi, M. et al., 1993; Russell, S. M. et al., 1993). Absence of the ⁇ chain leads to a severe combined immunodeficiency disease in humans (Noguchi, M. et al., 1993).
  • IL-2R ⁇ contains sequences from positions 288 to 321 homologous to the Src homology region 2 (SH2) that can bind to phosphotyrosine residues of some phosphoproteins.
  • SH2 Src homology region 2
  • Another molecule, designated pp97 has been suggested to be the tyrosine kinase physically associated with the IL-2R ⁇ chain (Michiel, D. F. et al., 1991).
  • IL-2 induced protein tyrosine kinase activity is due, at least in part, to activation of the p56 lck (lck), a src-family protein tyrosine kinase.
  • lck p56 lck
  • Controversy exists as to whether the serine/proline rich (Fung, M. R. et al., 1991) or an adjacent tyrosine rich “acidic” region (Hatakeyama, M. et al., 1991) of the IL-2R ⁇ chain is the lck binding site.
  • IL-2 also stimulates phosphorylation on serine residues of several proteins (Turner, B. et al., 1991; Valentine, M. V. et al., 1991).
  • Raf-1 a serine/threonine kinase, has been identified as a likely signal transducing element for several growth factor receptors (Carroll, M. P. et al., 1990; Morrison, D. K. et al., 1988; Baccarini, M. et al., 1991; Kovacina, K. S. et al., 1990; Blackshear, P. J. et al., 1990; App, H. et al., 1991).
  • the Raf-1 molecule has a molecular weight of 74 kD and can be divided into 2 functional domains, the amino-terminal regulatory half and the carboxy-terminal kinase domains (for review see Heidecker, G. et al., 1991).
  • Raf-1 has been identified as a crucial signal transducing element for ligand activated EPO receptors (Carroll, M. P. et al., 1991).
  • the IL-2R ⁇ chain and EPO receptors belong to the same family of receptors and share homologies within their cytoplasmic domains (D'Andrea, A. D. et al., 1989).
  • IL-2R Stimulation of the IL-2R results in the phosphorylation and activation of cytosolic Raf-1 serine/threonine kinase.
  • IL-2R stimulation leads to a 5 to 10 fold immediate/early induction of the c-raf-1 mRNA expression on freshly isolated, resting T cells (Zmuidzinas, A. et al., 1991) and results in up to a 12-fold increase in Raf-1 protein expression.
  • a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increases through G1.
  • Enzymatically active Raf-1 appears in the cytosol of IL-2 stimulated CTLL-2 cells (Hatakeyama, M. et al., 1991) and human T blasts (Zmuidzinas, A. et al., 1991). Following IL-2 stimulation, cytosolic Raf-1 molecules are phosphorylated on tyrosine and serine residues (Turner, B. et al., 1991). The laboratory of the present inventors have studied the signaling pathway by which IL-2 signals T cells to begin dividing. In these studies Raf-1 was identified in immunoprecipitates of the IL-2R ⁇ chain, suggesting that Raf-1 may be involved as an important element in IL-2 signaling.
  • Raf-1 molecules are physically associated with the IL-2R ⁇ chain and that following stimulation with IL-2, a protein tyrosine kinase phosphorylates Raf-1 thereby leading to translocation of Raf-1 from the IL-2 receptor into the cytosol (Maslinski, W. et al., 1992).
  • dissociation of enzymatically active Raf-1 from the IL-2R ⁇ chain, but not maintenance of IL-2R associated kinase activity is completely abolished by genistein, a potent tyrosine kinase inhibitor (Maslinski, W. et al., 1992).
  • IL-2 Prior to IL-2 stimulation, several serine, but not tyrosine nor threonine, residues of the IL-2R ⁇ chain are phosphorylated (Asao, H. et al., 1990). IL-2 induces rapid (i.e., within 10-30 min) phosphorylation of additional serines, tyrosines and threonines (Asao, H. et al., 1990; Hatakeyama, M. et al., 1991). Tyr 355 and Tyr 358 are major, but not exclusive, tyrosine phosphorylation sites of IL-2R (catalyzed by p561 lck in Vitro (Hatakeyama, M. et al., 1991)). The phosphorylation sites of the IL-2R ⁇ chain may play an important role in IL-2R ⁇ chain signal transduction and interactions with accessory molecules (like p561 lck and Raf-1).
  • Raf-1 Phosphorylation of Raf-1 has also been demonstrated in a human T cell line following CD4 cross-linking. Activation of Raf-1 has also been observed following TCR/CD3 complex stimulation by CD3 or Thy 1 cross-linking as well as an approximately four fold increase in c-raf-1 mRNA. In this case, Raf-1 phosphorylation occurs only on serines and is not observed if PKC had been down regulated. It is interesting to note in this context that GTPase-activating protein (GAP) activation and, consequently, Ras induction following TCR stimulation is also PKC mediated (Downward, J. et al., 1990).
  • GAP GTPase-activating protein
  • mice which lack IL-2 have shown that about 50% die by nine weeks of age (Schorle, H. et al., 1991). Although these mice appear to be phenotypically normal and can mount some cell-mediated responses (Kundig, T. M. et al., 1993), they ultimately develop inflammatory disease. Recently, it has been suggested that the reason the mice are still relatively normal is that there is an additional cytokine (IL-15) that signals through the IL-2 receptor ⁇ and ⁇ chains. Thus, there may be some compensation by IL-15 in these mice for the lack of the IL-2 molecules.
  • IL-15 additional cytokine
  • 14-3-3 appears to associate and interact with Raf-1 at multiple sites, i.e., amino terminal regulatory regions of Raf-1, kinase domain of Raf-1, zinc finger-like region of Raf-1, etc., with primary sites of interaction located in the amino-terminal regulatory domain (Fu et al., 1994; Freed et al., 1994).
  • cysteine- and serine-rich regions were found to be common elements and may be some of the determinants responsible for 14-3-3 binding (Morrison, 1994).
  • 14-3-3 constitutively associates with Raf-1 in vivo regardless of subcellular location or Raf-1 activation state or whether Raf-1 is bound to Ras (Fu et al., 1994; Freed et al., 1994), it is suggested that an alternate function of 14-3-3 may be a structural role in stabilizing the activity or conformation of signaling proteins (Morrison, 1994).
  • one of the aims of the present invention has been to determine the nature of interaction between the IL-2R ⁇ chain and Raf-1 and possibly other proteins or peptides involved in the IL-2- or IL-15-mediated intracellular processes. Accordingly, another aim of the present invention has been to find ways of inhibiting the binding between Raf-1 and IL-2R ⁇ , and between IL-2R ⁇ , 14-3-3 and other proteins directly involved in IL-2- or IL-15-mediated intracellular processes, and thereby provide a way in which autoimmune diseases in general, all graft rejection and graft-versus-host reactions may be treated successfully.
  • the present invention is based on the development of in vitro assay systems to determine the nature and specificity of the binding between Raf-1 and IL-2R ⁇ chain intracellular domain and the finding that the acidic region of the IL-2R ⁇ chain is essential for binding of Raf-1 to IL-2R ⁇ .
  • the binding of IL-2R ⁇ to Raf-1 is an essential step in the intracellular signaling process mediated by the IL-2R and IL-15R following IL-2/IL-15 stimulation, and is implicated, amongst others, in autoimmune diseases in general, allograft rejection and graft-versus-host reactions in particular.
  • the intracellular domain of the IL-2R ⁇ chain directly binds to Raf-1 and so-called 14-3-3 proteins.
  • the acidic domain of the intracellular domain of the IL-2R ⁇ chain that is homologous to the Ras effector domain, is critical for Raf-1 binding while the C-terminal portion of the intracellular domain of the IL-2R ⁇ chain interacts with 14-3-3 protein.
  • Raf-1 and 14-3-3 proteins form complexes on the IL-2R ⁇ chain intracellular domain and in the presence of enzymatically active p56 lck , but not p59 fYn , Raf-1/14-3-3 complexes dissociate from the intracellular domain of the IL-2R ⁇ chain.
  • Raf-1/14-3-3 proteins dissociate from the intracellular domain of the IL-2R ⁇ chain.
  • the present invention provides a compound capable of binding to Raf-1 protein, 14-3-3 proteins, or to the intracellular domain of the IL-2R ⁇ chain and being able to inhibit the binding of Raf-1 and/or 14-3-3 proteins to IL-2R ⁇ .
  • Embodiments of this aspect of the invention include: (i) A compound selected from proteins, peptides and analogs or derivatives thereof, and organic compounds; (ii) a compound being the 27 amino acid peptide corresponding to amino acid resides 370 to 396 of SEQ ID NO:2, derived from the acidic region of the mature human IL-2R ⁇ chain as set forth in FIG. 12 or analogs or derivatives thereof; (iii) a compound being selected from analogs of said 27 amino acid peptide in which one or more amino acid residues have been added, deleted or replaced, said analogs being capable of inhibiting the binding between Raf-1 and/or 14-3-3 and IL-2R ⁇ .
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention or a mixture of two or more thereof, as active ingredient and a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides an in vitro screening assay for isolating, identifying and characterizing compounds according to the invention, capable of binding to Raf-1, 14-3-3 proteins, or IL-2R ⁇ chain intracellular domain, comprising (a) providing a synthetically produced, a bacterially produced or a mammalian cell produced protein selected from IL-2R ⁇ chain protein or Raf-1 protein or 14-3-3 protein or portions of any one thereof, or mixtures of any of the foregoing; (b) contacting said protein of (a) with a test sample selected from prokaryotic or eukaryotic cell lysates, a solution containing naturally derived or chemically synthetized peptides, or a solution containing chemically synthetized organic compounds, to form a complex between said protein and said test sample; (c) isolating the complexes formed in (b); (d) separating the test sample from the protein in the complexes isolated in (c); and (e) analyzing said separated test sample of (d) to determine its
  • inventions include an in vitro screening assay wherein said assay is the herein described cell-free assay system; an in vitro screening assay wherein said assay is the herein described totally cell-free assay system; an in vitro assay for screening a compound capable of binding to Raf-1, and/or 14-3-3 proteins or IL-2R ⁇ intracellular domain and inhibiting the binding between Raf-1 and IL-2R ⁇ , said assay comprising the steps of determining the protein kinase reaction as described herein in Examples 1-6; as well as compounds isolated, identified and characterized by the in vitro assays according to the invention.
  • the present invention also provides: (i) compounds isolated, identified and characterized by any of the above in vitro assays; (ii) a pharmaceutical composition for the treatment of autoimmune diseases or graft-versus-host reactions containing a compound of the invention; and (iii) use of a compound of the invention for the treatment of autoimmune diseases transplant rejection or graft-versus-host reactions.
  • FIG. 1 depicts schematically the structure of the IL-2R ⁇ fusion proteins as described in Example 1;
  • FIG. 2 depicts the results illustrating the binding of Raf-1 from T-cell lysates to FLAG-HMK-IL-2R ⁇ chain related proteins as described in Example 1;
  • FIG. 3 depicts the results illustrating the interaction between bacterially derived (His) 6 -Raf-1 proteins with FLAG-HMK-IL-2R ⁇ chain related proteins as described in Example 2;
  • FIG. 4 depicts the results illustrating the products of protein kinase reaction performed on anti-FLAG beads coated with FLAG-HMK-IL-2R ⁇ chain proteins and exposed to T-cell lysates as described in Example 3;
  • FIG. 5 depicts the results illustrating the products of serine/threonine kinase reaction performed on anti-FLAG beads coated with FLAG-HMK-IL-2R ⁇ chain and exposed to T-cell lysates, as described in Example 3;
  • FIG. 6 depicts the results illustrating the products of protein kinase reaction performed on anti-FLAG beads coated with FLAG-HMK-IL-2R ⁇ chain related proteins and exposed to T-cell lysates, as described in Example 3;
  • FIGS. 7 ( a-c ) depict schematically the structure of the IL-2R ⁇ fusion proteins prepared for expression in mammalian (COS) cells (FIG. 7 a , IL-2R ⁇ chain contructs) and in bacterial cells (FIG. 7 b , FLAG-HMK-IL-2R ⁇ chain constructs), as well as the results of expression of these fusion proteins (FIG. 7 c ), as described in Example 4;
  • FIGS. 8 ( a-c ) depict the results illustrating the direct interaction between Raf-1 and 14-3-3 proteins with IL-2R ⁇ chain or portions thereof, as described in Example 4;
  • FIG. 9 depicts a schematic representation of the homology between IL-2R ⁇ chain (human) (amino acid residues 372 to 396 of SEQ ID NO:2) and the Ras (human) protein (SEQ ID NO:3), as described in Example 4;
  • FIGS. 10 ( a-b ) depict the results illustrating the abrogation by enzymatically active p56 lck of Raf-1 and 14-3-3 binding to the IL-2R ⁇ chain, as described in Example 4;
  • FIG. 10 c depicts the results illustrating the binding of Raf-1 and 14-3-3 proteins from T-cell lysates to the IL-2R ⁇ chain as described in Example 4.
  • FIG. 11 is a schematic illustration of the determination of the Raf-1/IL-2R ⁇ chain contact points as described in Example 5.
  • FIG. 12 is a schematic representation of the amino acid sequence of the human IL-2R ⁇ chain (SEQ ID NO:2), as described in Example 5.
  • the extracytoplasmic domain is in the upper part of the figure in upper case letters.
  • the peptide leader is indicated by lower case letters and the transmembrane region by underlined letters.
  • the acidic region (aa 313-382) is indicated by dashed underlined letters and the putative region (aa 345-371) involved in IL-2R ⁇ /Raf-1 interaction is shown by italic letters.
  • IL-2R ⁇ Chain Interaction with Raf-1 Proteins the IL-2R ⁇ Chain Region Involved in Raf-1 Binding
  • the direct interaction of the IL-2R ⁇ chain and Raf-1 binding has not been previously described. It has been widely believed that the IL-2R ⁇ mediated activation of Raf-1 involves the intermediacy of other proteins. In addition, it has not previously been determined whether or not 14-3-3 proteins ate capable of binding to the IL-2R ⁇ chain directly. Again, other intermediate proteins have been implicated in 14-3-3 binding. Furthermore, characterization of the proteins that are associated with the IL-2R ⁇ chain is limited by the low copy number of receptors per T cell (2-3 ⁇ 10 3 receptors/cell), complexity of the interactions between the receptor protein and the myriad of associated proteins.
  • a cell-free system in order to analyze the interaction between the IL-2R ⁇ chain and Raf-1 and/or 14-3-3 proteins, in particular, to identify the region(s) of the IL-2R ⁇ chain essential for binding to Raf-1 and/or 14-3-3 proteins.
  • the binding of the 14-3-3 proteins to IL-2R ⁇ is set forth in Example 4. This cell-free system was initially prepared as follows:
  • the IL-2R ⁇ chain cytoplasmic domain was cloned in a bacterial expression system and expressed as part of a fusion protein downstream from 17 hydrophilic amino acids comprising an antigenic epitope (FLAG) and a recognition site for heart muscle kinase (HMK) that permits in vitro radiolabeling of the fusion protein with [ ⁇ 32 P]-ATP and HMK (LeClair, K. P. et al., 1992; Blanar, M. A. and Rutter, R. J., 1992).
  • FLAG antigenic epitope
  • HMK heart muscle kinase
  • the FLAG-HMK-IL-2R ⁇ chain cytoplasmic domain expression plasmid was constructed by ligating the appropriate 1107 bp (NcoI-BamHI) cDNA fragment from the IL-2R ⁇ chain into the FLAG-HMK vector (LeClair et al., 1992; Blanar and Rutter, 1992) using synthetic linkers that facilitated cloning and maintain the proper translational frame.
  • BL-21 pLysS bacteria were transformed with the FLAG-HMK-IL-2R ⁇ construct, and protein expression was induced as described (LeClair et al., 1992).
  • the FLAG-HMK-IL-2R ⁇ chain cytoplasmic domain fusion protein was purified from bacterial lysate using the M2 anti-FLAG monoclonal antibody in a standard affinity chromatography procedure. More specifically, bacterial lysate proteins were absorbed onto anti-FLAG (M2) affinity column (IBI-Kodak, New Haven, Conn., USA). After washing the column, the adsorbed proteins were eluted with either glycine buffer (pH 3) or FLAG peptide (10 ⁇ 4 M).
  • Proteins in various fractions were analyzed for expected size (about 33 kDa) and purity after separation on SDS-PAGE and Commassie blue staining.
  • the presence of a functional HMK recognition site was confirmed by phosphorylation of the purified 33 kDa IL-2R ⁇ chain fusion protein by HMK.
  • the eluted fusion proteins were tested for susceptibility to phosphorylation by incubation with the catalytic subunit of bovine heart muscle kinase (Sigma) (1 U/ul) in buffer containing 20 mM Tric-HCl, pH 7.5, 1 mM DTT, 100 mM NaCl, 10 mM MgCl 2 and 1 ⁇ Ci [( ⁇ 32 P] ATP for 30 min at 37° C. followed by SDS-PAGE and autoradiography.
  • Purified FLAG-HMK-IL-2R ⁇ chain fusion proteins were used as an affinity reagent to probe for cytosolic proteins present in lysates of human T cells, metabolically labeled with [ 35 S]-methionine, that bind to the IL-2R ⁇ chain.
  • the human T cells being peripheral blood mononuclear cells were isolated using Ficoll-Hypaque, stimulated with phytohemagglutinin (5 ⁇ g/ml) in culture for 72 h, washed, maintained in culture for 3 days in the presence of IL-2 (10 U/ml), and then incubated without IL-2 for 24 hours.
  • the cells were suspended at 4 ⁇ 10 7 cells/ml at 37° C.
  • bacterially produced proteins (a) FLAG-HMK-IL-2R ⁇ chain wild type (WT); (b) FLAG-HMK-IL-2R ⁇ chain containing only the proline rich C-terminal (CT + ); FLAG-HMK-IL-2R ⁇ chain mutants lacking; (c) the serine rich region (S ⁇ ); (d) the acidic domain (A ⁇ ); (e) both acidic domain and proline rich C-terminal (A ⁇ CT ⁇ ); or (f) FLAG-HMK vector (v) which does not contain IL-2R ⁇ chain sequences (negative control), were absorbed on anti-FLAG affinity beads and washed.
  • WT FLAG-HMK-IL-2R ⁇ chain wild type
  • CT + proline rich C-terminal
  • S ⁇ the serine rich region
  • FIG. 2 is a reproduction of the relevant bands of an immunoblot of the above noted fusion proteins separated on SDS-PAGE, transferred to the Immobilon membrane and blotted with the anti-Raf-1 antibody. Relative band intensity is apparent from the immunoblot, and the calculated volume of each band corresponding to each different fusion protein is indicated below the band.
  • Raf-1 related fusion proteins i.e., FLAG-HMK-Raf-1 and (His) 6 -Raf-1 proteins were constructed, bacterially expressed and purified on affinity resins.
  • FLAG-HMK-Raf-1 expression plasmid For the construction of the FLAG-HMK-Raf-1 expression plasmid, PCR was performed using the Raf-1 cDNA as template and oligonucleotide primers designed to facilitate cloning into the FLAG-HMK-vector (for FLAG-HMK vector, see Example 1).
  • FLAG-HMK-Raf-1 protein was produced in BL-21 pLysS bacteria by IPTG induction, and purified on anti-FLAG affinity resin. Affinity purification yielded a 72-74 kD protein which was recognized by anti-Raf-1 antibody.
  • (His) 6 -Raf-1 expression plasmid PCR was performed using the Raf-1 cDNA as template and oligonucleotide primers designed to facilitate cloning into the pQE-30 plasmid according to the manufacturer's protocol (QIAGEN, QIAexpressionist; Chatsworth, Calif.).
  • (His) 6 -Raf-1 protein was produced in M15 bacteria by IPTG induction, and purified on Ni-NTA resin (QIAGEN). Affinity purification yielded a 72-74 kD protein which was recognized by anti Raf-1 antibody.
  • bacterially produced proteins FLAG-HMK-IL-2R ⁇ chain wild type (WT), FLAG-HMK-IL-2R ⁇ chain containing only proline rich C-terminal (CT + ), FLAG-HMK-IL-2R ⁇ chain mutants lacking:serine rich region (S ⁇ ), acidic domain (A ⁇ ), acidic domain and proline rich C-terminal (A ⁇ CT ⁇ ) were incubated with bacterially produced (His) 6 -Raf-1 (for all constructs see FIG. 1) followed by adsorption of FLAG-HMK-IL-2R ⁇ chain/Raf-1 complexes on anti-FLAG affinity beads.
  • IL-2R ⁇ chain/Raf-1 complexes were competitively eluted from anti-FLAG beads using buffer containing FLAG peptide. Eluted proteins were separated on SDS-PAGE, transferred onto Immobilon membrane and blotted with anti-Raf-1 antibody.
  • FIG. 3 is a reproduction of the relevant bands of an immunoblot of the above noted proteins separated on SDS-PAGE, transferred to the Immobilon membrane and blotted with the anti-Raf-1 antibody. Relative band intensity is apparent from the immunoblot and the calculated volume of each band corresponding to each different fusion protein is indicated below the band.
  • the two extreme right hand samples namely the second “WT” and the “VV” are positive and negative controls (see below) and the “+” and “ ⁇ ” signs indicate which IL-2R ⁇ -FLAG construct was reacted with Raf-1 protein.
  • FLAG-HMK-IL-2R ⁇ chain (WT) and deletion mutant lacking the serine rich region (S ⁇ ) of the IL-2R ⁇ chain bind Raf-1 proteins equally well.
  • mutants lacking the acidic domain of IL-2R ⁇ chain (A ⁇ ) express a significantly reduced capacity to bind Raf-1.
  • the amount of Raf-1 proteins bound to FLAG-HMK-IL-2R ⁇ A ⁇ mutant as estimated using Hewlett Packard ScanJet varied between 17% to 50% of the positive control value, i.e., 17-50% of Raf-1 binding to FLAG-HMK-IL-2R ⁇ chain WT.
  • Raf-1 serine/threonine kinase
  • Binding of the IL-2R ⁇ to the regulatory domain of Raf-1 may activate the kinase through a conformational change (Maslinski, W. et al., 1992).
  • a FLAG-HMK-Raf-1 fusion protein was constructed and expressed.
  • FLAG-HMK-IL-2R ⁇ chain wild type and deletional mutants lacking certain defined domains of the IL-2R ⁇ chain were used to identify IL-2R ⁇ chain domain involved in binding T-cell derived, active serine/threonine kinase. Assay conditions were similar to those noted above.
  • bacterially produced proteins FLAG-HMK-IL-2R ⁇ chain wild type (WT), FLAG-HMK-IL-2R ⁇ chain containing only proline rich C-terminal (CT + ), FLAG-HMK-IL-2R ⁇ chain mutants lacking:serine rich region (S ⁇ ), acidic domain (A ⁇ ), both acidic domain and proline rich C-terminal (A ⁇ CT ⁇ ), or FLAG-HMK vector which does not contain IL-2R ⁇ chain sequences (negative control) (for all constructs see diagram on FIG. 1) were absorbed on anti-FLAG affinity beads and washed. FLAG-HMK-fusion proteins coated beads were further used as affinity reagents to absorb proteins present in T-cell lysates.
  • T-cell derived proteins bound to FLAG-MHK fusion proteins were then tested for serine/threonine kinase activity in the absence or presence of exogenous substrates: Histone H-1 or (His) 6 -Mek-1.
  • Products of kinase reactions were boiled in SDS-PAGE sample buffer followed by separation on SDS-PAGE, transfer onto Immobilon membrane and autoradiography.
  • FIG. 4 is a reproduction of an autoradiogram of the products of the protein kinase reaction performed on anti-FLAG beads coated with the various IL-2R ⁇ fusion products, incubated with T cell lysates and subsequently subjected to SDS-PAGE and autoradiography.
  • FIG. 6 is a reproduction of an autoradiogram of the products of the kinase reaction performed, in the presence of (His) 6 -Mek-1 proteins, on various IL-2R ⁇ chain constructs exposed to T-cell lysates and then subjected to SDS-PAGE and autoradiography.
  • anti-FLAG affinity beads coated with FLAG-HMK-IL-2R ⁇ chain wild type (WT) or FLAG-HMK-IL-2R ⁇ chain mutant lacking serine-rich region (mutant S ⁇ ) and exposed to T-cell lysates retain active serine/threonine kinase that (i) phosphorylates p70 band which comigrates with Raf-1 proteins, and (ii) phosphorylates kinase inactive (His) 6 -Mek-1 proteins.
  • Bacterial lysates containing FLAG-HMK-IL-2R ⁇ chain wild type and (His) 6 -Raf-1 proteins were used (see Example 2) to test the hypothesis that the IL-2R ⁇ chain induces catalytic activity of Raf-1. Assay conditions were similar to those described above. Briefly, bacterially produced proteins: FLAG-HMK-IL-2R ⁇ chain wild type (WT) or FLAG-HMK (negative control) (for all constructs see FIG. 1) were incubated with bacterial lysates containing either (His) 6 -Raf-1 or (His), (negative control) followed by the absorption of protein complexes on anti-FLAG affinity beads.
  • Washed beads were tested for the presence of serine/threonine kinase activity in the presence of the exogenously added substrate, enzymatically inactive (His) 6 -Mek-1 kinase protein.
  • His enzymatically inactive
  • Products of kinase reactions were boiled in SDS-PAGE sample buffer followed by separation on SDS-PAGE, transfer onto Immobilon membrane and autoradiography.
  • IL-2R ⁇ Chain Interaction with Raf-1 and/or 14-3-3 Proteins the IL-2R ⁇ Chain Region Involved in Raf-1 and/or 14-3-3 Protein Binding
  • cDNAs encoding the IL-2R ⁇ chain or mutants lacking segments of its cytoplasmic domain were prepared and expressed in COS cells.
  • IL-2R ⁇ -box 1 ⁇ S ⁇ was made by cloning the full length wild type IL-2R ⁇ chain cDNA (SEQ ID NO:1) into the XbaI site of pBluescript II SK (Stratagene). This construct was then digested with NcoI-AflII. The NcoI/AflII sites were ligated with double stranded linker composed of oligonucleotides:
  • 5′TTAAGACCTTCTTCAGC3′ (antisense, bases 950-962, plus an AflII site; SEQ ID No:5).
  • This construct was then digested with XbaI and fragment containing sequences encoding IL-2R ⁇ chain was cloned back into pRcCMV.
  • pRcCMV-IL-2R ⁇ was digested with XbaI and cloned into XbaI site of pTZ19R (Pharmacia). This construct was then digested with NcoI-BstXI.
  • the 964 bp fragment containing sequences encoding most of the cytoplasmic domain of the IL-2R ⁇ chain was replaced with a 754 bp fragment obtained from NcoI and BstXI digestion of the AR(DRI)59/60 plasmid (Le Clair et al., 1992; Blanar et al., 1992) containing FLAG-HMK-IL-2R ⁇ -A ⁇ mutant encoding cDNA (see below).
  • the resultant pTZ-IL-2R ⁇ -A ⁇ plasmid contains sequences encoding an IL-2R ⁇ chain but lacking 210 bases encoding acidic domain was then digested with XbaI, and a fragment containing sequences encoding IL-2R ⁇ -A ⁇ was cloned back into pRcCMV.
  • FLAG-HMK-IL-2R ⁇ -S ⁇ mutant For the construction of FLAG-HMK-IL-2R ⁇ -S ⁇ mutant (serine-rich domain is deleted), a plasmid encoding FLAG-HMK-IL-2R WT was digested with Sac-AflII. After filling both ends, the plasmid was blunt end ligated.
  • FLAG-HMK-IL-2R ⁇ -A ⁇ mutant acidic domain is deleted
  • a 1048 bp fragment obtained from SacI-BamHI digestion of FLAG-HMK-IL-2R ⁇ -WT was further digested with PstI resulting in 3 fragments of 701, 210 and 136 bp. Fragments 701 and 136 were ligated back into the backbone of SacI-BamHI digested FLAG-HMK-IL-2R ⁇ -WT construct. The authenticity of each of the introduced mutations was confirmed by DNA sequence analysis.
  • FIG. 7 a there are shown schematic representations of the wild type (WT) and mutant (box 1 ⁇ S ⁇ ; A ⁇ ) IL-2R ⁇ chain protein constructs prepared as above for expression in COS cells.
  • FIG. 7 b there are shown schematic representations of the wild type (WTO and mutant (S ⁇ ; A ⁇ ) IL-2R ⁇ chain protein constructs prepared as above (see also Example 1, a(i) and (ii) above) for expression in COS cells.
  • constructs were introduced into COS cells and bacterial cells and the proteins were expressed, affinity purified from lysates of the cells, the purified proteins were separated on SDS-PAGE and stained with Commassie blue (for basic procedures see also Le Clair et al., 1992; Blanar and Rutter, 1992).
  • the procedure for expression of the constructs in bacterial cells followed by affinity purification, SDS-PAGE separation and Commassie blue staining has been described above (Exa example 1, a (i) and (ii)).
  • the procedure for expression of the constructs in COS cells followed by SDS-PAGE separation, affinity purification and Commassie staining was as follows:
  • COS cells were transfected via the DOTAP method (Boehringer-Mannheim, Indianapolis, Ind.) following the manufacturer's instructions.
  • the transfection cocktail contained 5 ⁇ g of DNA total and 30 ml of DOTAP in a final volume of 150 ml HBS (25 mM HEPES, pH 7.4 and 100 mM NaCl).
  • the COS cells were grown in DMEM medium supplemented with 10% heat-inactivated fetal calf serum, penicillin/streptomycin, 25 mM HEPES, pH 7.4, and L-glutamine.
  • the COS cells were exposed to the transfection cocktail for 12 hours, washed and subsequently cultured in fresh medium.
  • the transfected COS cells were lysed in 0.5 ml of lysis buffer on ice for 10 minutes, and subsequently centrifuged at 12,000 ⁇ g for 5 minutes, remaining supernatants were collected, and supplemented with pre-immune serum and protein G-agarose beads (BRL-Gibco, Gaithersburg, Md.), which had been previously washed in lysis buffer.
  • the samples were incubated at 4° C. for 30 minutes on a rocker. Supernatants were collected and supplemented with appropriate antibody, and later the protein G-agarose beads were added. Samples were washed 3 times for 15 min. each in lysis buffer and resuspended in Laemmli buffer and subsequently subjected to SDS-PAGE followed by Commassie blue staining for basic procedures (see also Maslinski, et al., 1992).
  • Antibodies used in the above affinity purification step included: a rabbit anti-serum raised against a 14-3-3 protein expressed in bacteria using standard procedures, this being a polyclonal anti-14-3-3 antibody protein; a rabbit anti-human 14-3-3 antibody that is cross-reactive with bacterial 14-3-3 proteins purchased from Upstate Biotechnology; an anti-Raf-1 (C1) antibody purchased from Santa Cruz Biotechnology; an anti-human IL-2R ⁇ antibody called Mik-ol (as described in Tsudo et al, 1989 and obtained from M Tsudo, Kyoto, Japan).
  • FIG. 7 ( c ) there is shown a reproduction of the relevant bands of a Commassie blue stained, SDS-PAGE separation of affinity purified FLAG-HMK-IL-2R ⁇ chain related (wild type and mutant) fusion proteins which were expressed in the COS cells.
  • COS cells were transfected, as set forth hereinabove, with constructs encoding full-length or deletional mutants of the human IL-2R ⁇ chain, immunoprecipitated with an anti-IL-2R ⁇ chain antibody Mik- ⁇ 1 (see (ii) above) and blotted with anti-Raf-1 or anti-14-3-3 antibodies (see (ii) above).
  • lysates of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells were passed through anti-FLAG affinity beads containing purified FLAG-HMK-IL-2R ⁇ related proteins (see (i) and (ii) above, as well as Examples 1-3).
  • the absorbed proteins were washed, eluted with FLAG peptide and probed for the presence of Raf-1 and 14-3-3 proteins on immunoblots.
  • peripheral blood mononuclear cells were isolated using Ficoll-Hypaque (Pharmacia), stimulated with PHA (Sigma) 5 mg/ml in culture for 72 hours, washed, maintained in culture for 3 days in the presence of IL-2 (Hoffman-La Roche) 10 U/ml, and then incubated without IL-2 for 24 hours. Washed cells (about 4 ⁇ 10 7 ) were lysed in Dounce homogenization buffer, centrifuged (15 ⁇ 10 3 ⁇ g for 15 min.) and supernatants applied onto washed anti-FLAG (M2) affinity column (IBI-Kodak) coated with bacterial lysates interacted with one of the FLAG-HMK fusion proteins.
  • M2 washed anti-FLAG affinity column
  • proteins adsorbed onto the anti-FLAG affinity column were eluted with the same buffer supplemented with FLAG peptide (10 ⁇ 4 M) and subjected to SDS-PAGE and immunoblotting described hereinabove.
  • FIGS. 8 a-c The results of the above experiments are shown in FIGS. 8 a-c:
  • FIG. 8 a there is shown a reproduction of immunoblots performed on lysates from transfected COS cells which were transfected with the various constructs IL-2R ⁇ -WT, IL-2R ⁇ -box 1 ⁇ S ⁇ , IL-2R ⁇ -A ⁇ , or, as a control, a vector having no IL-2R ⁇ construct (vector).
  • the COS cell lysates were immunoprecipitated with anti-Raf-1 or anti-14-3-3 antibodies. From the results shown in FIG. 8 a it is apparent that both IL-2R ⁇ -WT and the IL-2R ⁇ -box 1 ⁇ S ⁇ mutant bound both Raf-1 and 14-3-3 proteins. In contrast, the IL-2R ⁇ chain A ⁇ mutant failed to bind Raf-1 and bound only 14-3-3 proteins.
  • FIG. 8 b there is shown a reproduction of an immunoblot performed on lysates from PHA activated peripheral blood mononuclear cells, which were passed through anti-FLAG affinity beads containing purified FLAG-HMK-IL-2R ⁇ related proteins. The adsorbed proteins were washed, eluted with FLAG peptide and probed for the presence of Raf-1 and 14-3-3 on immunoblots. From the results shown in FIG. 8 b , it is apparent that the same specific interactions (as in FIG.
  • T-cell lysates were passed through IL-2R ⁇ chain-derived affinity columns, i.e., IL-2R ⁇ -WT and the IL-2R ⁇ -box 1 ⁇ S ⁇ mutant but not the IL-2R ⁇ chain A ⁇ mutant bound to Raf-1.
  • the Raf-1 protein is at basal levels as shown by phosphorylation of exogenously added kinase inactive MEK protein (see Examples 2 and 3 above).
  • the proteins bound to the beads were washed, eluted with FLAG peptide and probed by immunoblotting. Since the bacterial lysates contained a 28 kD protein, immunoreactive with antibody raised against a highly conserved region of the 14-3-3 protein (residues 119-129 of human 14-3-3) no attempt was made to co-express human 14-3-3 proteins. As is apparent from FIG. 8 c , there is direct binding between Raf-1 and the acidic region of the IL-2R ⁇ chain. Further, as in COS cells, 14-3-3 proteins present in bacterial lysates bound directly to the C-terminal portion of the IL-2R ⁇ chain. Thus, it appears that the homology between mammalian and bacterial 14-3-3 proteins is sufficient to preserve the 14-3-3 binding site to Raf-1 and the IL-2R ⁇ chain.
  • Raf-1 plays a central role in these three molecular interactions (Raf-1—14-3-3 - IL-2R ⁇ ).
  • This notion is further supported by the observation that the A ⁇ region of the IL-2R ⁇ chain is homologous to the effector domain or Ras and Rap1A that binds to Raf-1 (see, for example, Zhang et al., 1993; Nassar et al., 1995).
  • the homology between Ras (H-Ras) and the A region of IL-2R ⁇ is depicted schematically in FIG. 9 .
  • the interaction between IL-2R ⁇ chain (amino acids 371-395) may therefore be a key factor in Raf-1 immobilization through the IL-2R ⁇ chain at the plasma membrane.
  • FIG. 10 a is a reproduction of the above immunoblot. From these results it is apparent that co-transfection of COS cells with the IL-2R ⁇ chain and p56 lck resulted in the abrogation of Raf-1/14-3-3 binding to the IL-2Rb chain. In contrast, another src-like kinase, p59 fyn did not cause this dissociation. In addition, a study of the dissociation of pre-formed IL-2R ⁇ chain/Raf-1/14-3-3 complexes by enzymatically active p56 lck was also carried out.
  • the IL-2R ⁇ chain may therefore bypass the requirement for Ras activation in the membrane localization of Raf-1 (see Leevers et al., 1994; Stokoe et al., 1994).
  • Two distinct regions of the IL-2R ⁇ chain involved in the optimal binding of Raf-1 and 14-3-3 proteins may enable “permissive” Raf-1 binding and activation, i.e., the IL-2R ⁇ chain mutant lacking A-region may bind some of Raf-1 proteins through the binding to 14-3-3 proteins associated with C-terminal part (14-3-3 binding domain) of the receptor.
  • BAF cells expressing the mutant IL-2R ⁇ chain lacking the A-region still respond to IL-2 albeit more weakly than those expressing the wild-type molecule (Hatakeyama et al., 1989).
  • Raf-1 activation occurring in the absence of the IL-2R ⁇ A domain may be achieved via IL-2 induced activation of Ras (see for example, Izquierdo-Pastor et al., 1995).
  • the same portion of the IL-2R ⁇ chain (acidic domain) is needed for activation of the Raf-1 enzymatic activity (the so-called protein kinase activity).
  • the above acidic domain of the IL-2R ⁇ chain that is homologous to the Ras effector domain, is critical for Raf-1 binding, it is the C-terminal portion of the receptor which interacts with 14-3-3 proteins.
  • Raf-1/14-3-3 complexes dissociate from the IL-2R ⁇ chain, an event directly related to IL-2 mediated activation of IL-2R and subsequent intracellular signalling.
  • Two in vitro binding assays have been developed which are suitable for screening a number of samples for the presence of compounds or substances which have blocking activity, i.e., that are capable of blocking the binding or interaction of the IL-2R ⁇ chain to Raf-1, and thereby blocking the signaling pathway initiated by IL-2/IL-15 binding to its receptor (see Examples 5 and 6 below).
  • Such compounds or substances would thereby be useful for the treatment of autoimmune diseases in general, transplant rejection and graft-versus-host rejection process in particular, by being able to block the IL-2/IL-15-mediated signaling pathway.
  • the first such assay is a cell-free system in which bacterially produced or mammalian cell (COS cells) produced IL-2R ⁇ chain fusion proteins are interacted with T cell lysates to isolate, identify and characterize compounds, for example, Raf-1 protein, and 14-3-3 proteins capable of binding specifically to the IL-2R ⁇ chain intracellular domain or portions thereof.
  • COS cells bacterially produced or mammalian cell
  • IL-2R ⁇ chain fusion proteins are interacted with T cell lysates to isolate, identify and characterize compounds, for example, Raf-1 protein, and 14-3-3 proteins capable of binding specifically to the IL-2R ⁇ chain intracellular domain or portions thereof.
  • the second such assay is the so-called totally cell-free system in which bacterially produced or mammalian cell produced IL-2R ⁇ chain fusion proteins are interacted with bacterially produced Raf-1 protein ((His) 6 -Raf-1) and 14-3-3 proteins to isolate, identify and characterize the nature of the binding between the IL-2R ⁇ chain intracellular domain or portions thereof and the Raf-1 and 14-3-3 proteins.
  • Raf-1 protein (His) 6 -Raf-1) and 14-3-3 proteins
  • protein kinase reaction which occurs following the binding of Raf-1 and 14-3-3 proteins to a specific region of the intracellular domain of IL-2R ⁇ (the acidic domain and the acidic and proline-rich C-terminal region). This determination of the protein kinase reaction is an indicator of the initiation of the intracellular signaling process which is apparently initiated by the binding of Raf-1 and/or 14-3-3 to IL-2R ⁇ .
  • the determination of the protein kinase activity in vitro provides a reliable assay means for determining whether or not another compound, for example, peptides, organic compounds, etc., are capable of disrupting the binding between Raf-1 and 14-3-3 proteins and IL2-R ⁇ and thereby inhibiting the kinase reaction which is essential to the intracellular signaling mediated by IL-2R.
  • another compound for example, peptides, organic compounds, etc.
  • bacterially produced or mammalian cell produced IL-2R ⁇ chain intracellular domain (WT) and/or IL-2R ⁇ chain intracellular domain analogs such as those containing only the acidic domain or containing both the acidic and proline-rich C terminal domains may be employed as the substrate to which will be exposed samples containing the peptides, organic compounds, etc., which are to be screened to isolate those which specifically bind the IL-2R ⁇ chain.
  • WT mammalian cell
  • IL-2R ⁇ chain intracellular domain WT
  • IL-2R ⁇ chain intracellular domain analogs such as those containing only the acidic domain or containing both the acidic and proline-rich C terminal domains
  • WT IL-2R ⁇ chain intracellular domain
  • analogs such as those containing only the acidic domain or containing both the acidic and proline-rich C terminal domains
  • the acidic region of the IL-2R ⁇ chain is the region responsible for direct binding to Raf-1 and the C-terminal region is responsible for direct binding to 14-3-3 proteins.
  • the acidic region encompasses amino acids 313-382 of the mature human IL-2R ⁇ chain.
  • Raf-1 and 14-3-3 also form complexes and appear to bind IL-2R ⁇ and to dissociate therefrom in the form of complexes.
  • the proline-rich C-terminal portion of the IL-2R ⁇ chain (amino acids 383-525) is not critical for Raf-1 binding, but is critical for 14-3-3 binding; this portion of the IL-2R ⁇ chain may at most stabilize Raf-1 binding via the binding of 14-3-3 at this region which is complexed to Raf-1.
  • FIG. 11 there is shown a scheme of the essential portions of the IL-2R ⁇ intracellular domain (intracytoplasmic region) that are involved in binding to Raf-1 and are thus directly involved in the IL-2R mediated intracellular signaling.
  • FIG. 12 there is shown, schematically, the amino acid sequence of the human IL-2R ⁇ chain.
  • the extra cytoplasmic domain is in the upper part of the figure (capital letters); the peptide leader region is indicated by lower letters and the transmembrane region is indicated by underlined letters; and the intracytoplasmic domain is shown in the lower part of the figure, in which the acidic region (a.a. 313-382) is indicated by dotted underlined letters within which region (a.a. 345-371) are shown by italic capital letters the amino acid residues involved directly in IL-2R ⁇ interaction, of which residues those shown by bold capital italic letters are the acidic residues.
  • the serine residues of the serine-rich region in the intracytoplasmic domain are indicated by crossed-out capital S letters.
  • One such peptide which is likely to be capable of disrupting the binding between Raf-1 and IL-2R ⁇ and between Raf-1/14-3-3 and IL-2R ⁇ is a 27 amino acid peptide derived from analysis of deletion mutants (see Examples 1-4 above), being part of the acidic domain and having a sequence corresponding to amino acids 345-371 of the mature IL-2R ⁇ chain protein (i.e., peptide having amino acid residues corresponding to amino acids 370 to 396 of SEQ ID No:2, see FIG. 12 ).
  • Analogs of the above 27 amino acid peptide will be made by standard chemical synthesis procedures well known in the art or by standard recombinant DNA techniques. Such analogs will include those having one or more amino acids deleted, added or replaced with respect to above 27 amino acid peptide and which will be characterized by their ability to inhibit the binding between Raf-1 and/or 14-3-3 proteins and IL-2R ⁇ .
  • proteins or peptides which are likely to be capable of specifically binding to Raf-1 and/or IL-2R ⁇ and which may be capable of inhibiting the binding between Raf-1 and/or 14-3-3 proteins and IL-2R ⁇ include one or more proteins derived from the 14-3-3 family of proteins or specific peptides derived therefrom or any analogs, derivatives thereof.
  • organic compounds with some lipophilic characteristics may be most useful in view of the fact that in practice, such compounds to be used pharmaceutically would have to have the ability to pass through the cell membrane.
  • peptides can be chemically modified or derivatized to enhance their permeability across the cell membrane and facilitate the transport of such peptides through the membrane and into the cytoplasm.
  • Muranishi et al. (1991) reported derivatizing thyrotropin-releasing hormone with lauric acid to form a lipophilic lauroyl derivative with good penetration characteristics across cell membranes. Zacharia et al.
  • the compounds of the present invention which are capable of inhibiting the binding of Raf-1 and/or 14-3-3 proteins to the cytoplasmic domain of IL-2R ⁇ , can be conjugated or complexed with molecules that facilitate entry into the cell.
  • U.S. Pat. No. 5,149,782 discloses conjugating a molecule to be transported across the cell membrane with a membrane blending agent such as fusogenic polypeptides, ion-channel forming polypeptides, other membrane polypeptides, and long chain fatty acids, e.g., myristic acid, palmitic acid.
  • a membrane blending agent such as fusogenic polypeptides, ion-channel forming polypeptides, other membrane polypeptides, and long chain fatty acids, e.g., myristic acid, palmitic acid.
  • nutrient receptors such as receptors for biotin and folate
  • a complex formed between a compound to be delivered into the cytoplasm and a ligand, such as biotin or folate is contacted with a cell membrane bearing biotin or folate receptors to initiate the receptor mediated trans-membrane transport mechanism and thereby permit entry of the desired compound into the cell.
  • screening directed at small peptides is also advantageous to isolate and develop more stable peptidomimetic-type drugs.
  • small peptides e.g., that noted above (having between 20-30 amino acid)
  • screening directed at small peptides is also advantageous to isolate and develop more stable peptidomimetic-type drugs.
  • peptides in accordance with the invention may be any peptide of natural origin isolated in the above in vitro screening assays or may be any peptide produced by standard peptide synthesis procedures. Suitable peptides are those capable of interfering with the interaction between Raf-1 and/or 14-3-3 proteins with IL-2R ⁇ and thereby inhibiting the intracellular signalling process mediated by IL-2R ⁇ .
  • organic compounds in accordance with the present invention may be any known pharmaceutically utilized compound or any newly synthetized compound prepared by standard chemical synthesis methods. Suitable such compounds are those capable of interfering with the interaction between Raf-1 and/or 14-3-3 proteins with IL-1 2R ⁇ and thereby inhibiting the intracellular signalling process mediated by IL-2R ⁇ .
  • compositions of the invention may thus be used as the active ingredients in pharmaceutical compositions for the treatment of autoimmune diseases in general, or host-versus-graft reactions in particular.
  • pharmaceutical compositions of the invention are those comprising a pharmaceutically acceptable carrier, stabilizer or excipient and the above active ingredients of the invention.
  • compositions may be formulated in any acceptable way to meet the needs of the mode of administration. Any accepted mode of administration can be used and determined by those skilled in the art.
  • administration may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the pharmaceutical composition of the present invention may be administered alone or in conjunction with other therapeutics directed to the condition, or directed to other symptoms of the condition.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art, and can be prepared according to routine methods.
  • compositions comprising the inhibitory compounds of the present invention include all compositions wherein the inhibitory compound is contained in an amount effective to achieve its intended purpose.
  • the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspension of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions that may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. optionally, the suspension may also contain stabilizers.
  • compositions include suitable solutions for administration by injection, and contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active component (i.e., compounds that inhibit the binding of Raf-1 or 14-3-3 proteins to IL-2R ⁇ ) together with the excipient.
  • active component i.e., compounds that inhibit the binding of Raf-1 or 14-3-3 proteins to IL-2R ⁇
  • compositions which can be administered rectally include suppositories.

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US09/194,145 1996-05-23 1997-05-22 Compounds that inhibit the binding of RAF-1 or 14-3-3 proteins to the beta chain of IL-2 receptor, and pharmaceutical compositions containing same Expired - Fee Related US6281193B1 (en)

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PCT/US1997/008542 WO1997044058A1 (fr) 1996-05-23 1997-05-22 Composes inhibant la liaison de proteines raf-1 ou 14-3-3 a la chaine beta du recepteur d'il-2, et compositions pharmaceutiques les contenant

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070161680A1 (en) * 2005-08-30 2007-07-12 Novartis Ag Substituted benzimidazoles and methods of their use
US20100004253A1 (en) * 2006-09-19 2010-01-07 Novartis Ag Biomarkers of target modulation, efficacy, diagnosis and/or prognosis for raf inhibitors
WO2010100127A1 (fr) 2009-03-04 2010-09-10 Novartis Ag Dérivés d'imidazole disubstitués en tant que modulateurs de la protéine kinase raf
WO2011025927A1 (fr) 2009-08-28 2011-03-03 Irm Llc Composés et compositions en tant qu'inhibiteurs de protéine kinase
WO2011023773A1 (fr) 2009-08-28 2011-03-03 Novartis Ag Composés et compositions inhibiteurs de protéines kinases
WO2011097526A1 (fr) 2010-02-05 2011-08-11 Irm Llc Composés et compositions comme inhibiteurs de protéine-kinases
WO2011161216A1 (fr) 2010-06-25 2011-12-29 Novartis Ag Composés et compositions d'hétéroaryle en tant qu'inhibiteurs de protéine kinases
US10690489B2 (en) 2011-11-23 2020-06-23 The Trustees Of Columbia University In The City Of New York Systems, methods, and media for performing shape measurement

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009544617A (ja) * 2006-07-21 2009-12-17 ノバルティス アクチエンゲゼルシャフト ベンゾイミダゾリルピリジルエーテルの製剤
WO2022125694A1 (fr) * 2020-12-09 2022-06-16 Asher Biotherapeutics, Inc. Fusions de polypeptides d'interleukine avec des molécules de liaison à l'antigène bispécifique pour moduler la fonction de cellules immunitaires
WO2024050383A2 (fr) * 2022-08-29 2024-03-07 The Regents Of The University Of California Biocapteurs de ras

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0408790A1 (fr) * 1989-07-20 1991-01-23 Taniguchi, Tadatsugu, Dr. Récepteur protéique recombinant

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Hatakeyama et al. Science 244:551-556, 1989.*
Janeway et al. Immunobiology: The Immune System in Health and Disease, 3rd Edition, pp. 10:22 and 12:19, 1997.*
Kawamoto, et al, Cytotechnology 17:103-108, 1995.*
Kolch et al., Oncogene 5:713-720, 1990.*
Liblau et al., Immunology Today, 18:599-604, 1997.*
Ngo et al., The Protein Folding Problem and Tertiary Structure Prediction, Merz et al (ed) Birkhauser, Boston MA pp. 433 and 492-495, 1994.*
Pang et al Blood 80:724-732, 1992.*
Paul, William, Fundamental Immunology, 4th Edition, Lippincott-Raven, Philadelphia, PA, 1999. *
Skoulakis et al. Neuron 17 (5):931-44, 1996.*

Cited By (16)

* Cited by examiner, † Cited by third party
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US20100256375A1 (en) * 2005-08-30 2010-10-07 Novartis Vaccines And Diagnostics, Inc. Substituted benzimidazoles and methods of preparation
US8592459B2 (en) 2005-08-30 2013-11-26 Novartis Ag Substituted benzimidazoles and methods of their use
US7482367B2 (en) 2005-08-30 2009-01-27 Novartis Vaccines And Diagnostics, Inc. Substituted benzimidazoles and methods of their use
US20070161680A1 (en) * 2005-08-30 2007-07-12 Novartis Ag Substituted benzimidazoles and methods of their use
US7767820B2 (en) 2005-08-30 2010-08-03 Novartis Vaccines And Diagnostics, Inc. Substituted benzimidazoles and methods of preparation
US20080287682A1 (en) * 2005-08-30 2008-11-20 Novartis Ag Substituted benzimidazoles and methods of preparation
US20100234394A1 (en) * 2005-08-30 2010-09-16 Novartis Vaccines And Diagnostics, Inc. Substituted benzimidazoles and methods of their use
US7732465B2 (en) 2005-08-30 2010-06-08 Novartis Vaccines And Diagnostics, Inc. Substituted benzimidazoles and methods of their use
US20100004253A1 (en) * 2006-09-19 2010-01-07 Novartis Ag Biomarkers of target modulation, efficacy, diagnosis and/or prognosis for raf inhibitors
WO2010100127A1 (fr) 2009-03-04 2010-09-10 Novartis Ag Dérivés d'imidazole disubstitués en tant que modulateurs de la protéine kinase raf
WO2011023773A1 (fr) 2009-08-28 2011-03-03 Novartis Ag Composés et compositions inhibiteurs de protéines kinases
WO2011025927A1 (fr) 2009-08-28 2011-03-03 Irm Llc Composés et compositions en tant qu'inhibiteurs de protéine kinase
EP2727918A1 (fr) 2009-08-28 2014-05-07 Irm Llc Composés et compositions en tant qu'inhibiteurs de protéine kinase
WO2011097526A1 (fr) 2010-02-05 2011-08-11 Irm Llc Composés et compositions comme inhibiteurs de protéine-kinases
WO2011161216A1 (fr) 2010-06-25 2011-12-29 Novartis Ag Composés et compositions d'hétéroaryle en tant qu'inhibiteurs de protéine kinases
US10690489B2 (en) 2011-11-23 2020-06-23 The Trustees Of Columbia University In The City Of New York Systems, methods, and media for performing shape measurement

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AU3133997A (en) 1997-12-09
WO1997044058A1 (fr) 1997-11-27
IL127210A0 (en) 1999-09-22
EP0910401A1 (fr) 1999-04-28
JP2000511419A (ja) 2000-09-05
CA2256109A1 (fr) 1997-11-27
AU728701B2 (en) 2001-01-18
US20010016194A1 (en) 2001-08-23

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